Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that causes reproductive and respiratory disease in swine. PRRS was first recognized in midwestern United States in the late 1980s.[1] Pork producers and veterinarians use vaccination, test-and-removal, and other farm-management practices as strategies to control spread of the virus. Although clinical signs of PRRSV infection are now well known, a number of other common respiratory and reproductive diseases may appear clinically similar which has made laboratory diagnostic testing important. Testing populations of pigs for presence of the virus requires taking blood or oral fluid[2] samples and sending them to a diagnostic lab.
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A brief overview below of PRRS diagnostic tests focuses only on tests which detect the virus itself or its genetic material from ante-mortem samples (from live animals). More comprehensive discussions of PRRS tests exist, which include tests that detect antibody to the virus.[3]
Laboratory-based diagnostic tests have evolved significantly since initial discovery of the PRRS virus in the late 1980s. Initially viral culture was used to confirm PRRSV in serum or tissue samples. This process involves growing the virus in-vitro on cell lines over a period of 3–14 days or longer. If cytopathic effect is observed during culture, the culture is confirmed as the PRRS virus by direct fluorescent antibody or other confirmation method prior to reporting the sample as positive for presence of PRRSV.
Polymerase chain reaction (PCR) assays came into use in the mid to late 1990s. Reduced turnaround time (1–2 days) and improved sensitivity were gained from applying this new technology and it quickly became the predominant means of antemortem diagnosis as well as confirmation of viral culture. PCR was originally developed for DNA targets but the application of reverse-transcriptase enzymes RT-PCR allowed the method to extend to RNA targets.
Nested PCR is a 2-step approach which targets outer and inner portions of a sequence of DNA. Initial PCR assays used by diagnostic labs in the late 1990s tended to be nested assays as they showed improved sensitivity over non-nested PCR.[4] Because the nested PCR requires handling of amplified PCR products during testing, it suffers from false-positives if not done carefully.
The innovation of fluorescent-labeled DNA probes and high-throughput instrumentation to read them brought Real-time PCR to diagnostic labs. Real-time PCR assays offered as-good or better sensitivity than nested PCR, fast turnaround time in the lab, and lower rates of cross-contamination via closed-tube amplification. The specific chemistry used in diagnosis of PRRSV is commonly Taqman probes which emit light when they bind to the target sequence. Taqman probes focus on a small, roughly 100 base-pair, fragment of the PRRS genome.
The RNA of any given RNA virus changes over time as the virus mutates due (in part) to the infidelity of the RNA polymerase it uses to replicate.[1] As an RNA virus with a 15 kb genome, PRRS mutates at a relatively high rate as it is transmitted from pig-pig over time.[5] The calculated rate of PRRSV nucleotide substitution is the highest reported so far for an RNA virus. It is estimated as 4.7-9.8 x 10-2 / site / year.[1]
Though the real-time PCR tests used now have high sensitivity and specificity, these improvements have come with some hazards as well. Real-time PCR using Taq-man chemistry is prone to false-negative results when the virus mutates.[6][7][8] A false negative result occurs when a test fails to detect the presence of the virus. Studies have found that even a single base-pair change in the viral RNA under the labeled probe can cause failure of detection.[6] This specific source of the false-negative is not due to operator error on the part of the lab and is un-knowable at the time of testing.
The scenario that follows demonstrates how this hazard can result in risk to pork producers and laboratories:
→A strain of PRRS virus mutates during circulation within a herd. This strain spreads and becomes the predominant strain within the herd.
This series of events is a frustrating and expensive event for veterinarian, diagnostic lab, and animal owners. Many labs in the United States each use their own real-time PCR method and communication of test failures due to new strains to other diagnostic labs is difficult. As a result, information learned about new strains is not leveraged across many diagnostic labs. Due to the cost of testing and rapid detection of new virus introduction, PCR alone is often relied on as the primary screening tool. This over-reliance on a single diagnostic assay (of which none are 100% sensitive and specific) lead to longer interval of virus spread while the problem is being resolved.
Veterinarians can reduce the impact of this risk by paying close attention to clinical signs and utilizing more than one PRRS diagnostic test. Early communication with the lab is essential as often other methods can quickly be employed on existing samples. Given the rate of mutation for the PRRS virus, contingency plans should be developed for false-negative events that include selection of alternative labs and tests.
Some laboratories have moved to the use of commercially-developed and maintained real-time PCR assays, which transfers the work of assay updates to a 3rd party albeit at a significant extra cost over in-house developed assays. In recent years, this strategy has allowed quicker response to new variants than would have been previously possible (unpublished). By commercial manufacturers leveraging assay updates across multiple labs, it is possible that detection capabilities for all client labs is improved. The flip-side of this approach is that if all labs run the same assay, there are limited options for veterinarians when an alternate assay is quickly needed.
Earlier technologies such as nested PCR are often called on during an investigation if the lab has retained the capability to perform them. By using these earlier methods the laboratory staff are more quickly able to identify the new strain due to their more robust detection capabilities.